Remedies for lung cancer

ABSTRACT

By using a combination of amrubicin or a pharmaceutically acceptable salt thereof with another medicament for treating lung cancer (irinotecan, vinorelbine, gemcitabine, etc.), it is intended to provide medicaments for treating lung cancer having an improved antitumor therapeutic effect on lung cancer and reduced side effects. Lung cancer can be effectively treated using these medicaments.

TECHNICAL FIELD

The present invention relates to a medicament for treating lung cancer,comprising amrubicin or a pharmaceutically acceptable salt thereof as anactive ingredient, for a use in combination with another medicament fortreating lung cancer.

BACKGROUND ART

Amrubicin((+)-(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-D-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenedione)is an anthracycline compound represented by the following chemicalstructural formula (JP 3-5397B, U.S. Pat. No. 4,673,668):

Amrubicin is easily reduced in vivo to form a metabolite (amrubicinol)which is a 13-hydroxylated product. This amrubicinol has a considerablystronger inhibitory activity against growth of tumor cells than that ofamrubicin. Doxorubicin and daunomycin, the other anthracyclinecompounds, also form reduced metabolites, which in contrast have reducedactivities (Cancer Chemothr. Pharmacol., 30, 51-57 (1992)). Also for thecardiac toxicity, amrubicin is far weaker than doxorubicin in rabbitchronic experimental model (Invest. New Drug, 15, 219-225 (1997)).

It has been known that, although anthracycline compounds have similarstructures, they are different in their indications, action mechanismsand so on as described below. Daunorubicin and idarubicin are approvedfor treatment of leukemia but are not approved for treatment of solidcancers. On the other hand, doxorubicin, epirubicin, pirarubicin andaclarubicin are approved for treatment of solid cancers. Daunorubicinand doxorubicin inhibit synthesis of DNA and synthesis of RNA in similardegrees but aclarubicin and marcellomycin inhibit synthesis of RNA morestrongly than synthesis of DNA; therefore their mechanism of exertingantitumor activities are quite different (JJSHP, 27, 1087-1110 (1991)).It has been known that, in this manner, even if drugs belong to the samecategory of anthracycline, they have different effect depending on thekind of cancer, and the same anticancer agent has different effectdepending on the kind of cancer. Consequently, it is necessary tospecifically confirm by experiment whether or not a specific anticanceragent is effective against a specific tumor (cancer).

It has been described that a use of amrubicin hydrochloride incombination with cisplatin or the like exhibits an additive effect onhuman T-cell leukemia MOLT-3 cell strain and human osteosarcoma MG-63cell strain (Investigational New Drugs, 14, 357-363 (1996)).Additionally, an effect has been described, in an experiment with murineleukemia P388 cell strain, for a combined use of amrubicin and cisplatinin vivo (Yoshikazu YANAGI et al., Abstracts of publications in JapaneseCancer Association, No. 2168 (1989)). No report, however, has beendescribed for a combined use of amrubicin and another medicament fortreating lung cancer against lung cancer and for side effects.

DISCLOSURE OF INVENTION

The problem to be solved by the present invention is to provide amedicament for treating lung cancer with increasing antitumortherapeutic effect and reducing side effects.

As the result of extensive studies, the present inventors have found thefacts that lung cancers can be remarkably cured without increasing sideeffects observed in the single use of agents when amrubicin and anothermedicament for treating lung cancer are used in combination, and thatthe side effects can be dramatically reduced when respective doses aredecreased keeping the therapeutic effect in the combined use ofamrubicin and another medicament for treating lung cancer. Furthermore,the present inventors have also found that the expression level ofcarbonyl reductase 1 gene, which product can convert amrubicin into itsactive metabolite amrubicinol, is higher in the cells of lung cancersuch as small cell lung cancer, lung adenocarcinoma, squamous cell lungcarcinoma and large cell lung carcinoma than in normal lung cells andleukemic cells. Accordingly, they have found that amrubicin isselectively changed into amrubicinol and works in the cells of lungcancer more than in normal lung cells or leukemic cells, and thus theside effects will be reduced by the present invention.

The present invention has been completed according to the aboveknowledge.

Namely, the present invention is as follows:

[1] A medicament for treating lung cancer, comprising amrubicin or apharmaceutically acceptable salt thereof as an active ingredient, for ause in combination with another medicament for treating lung cancer.

[2] The medicament for treating lung cancer as described in [1], whereinthe lung cancer is small cell lung cancer, lung adenocarcinoma, squamouscell lung carcinoma or large cell lung carcinoma.

[3] The medicament for treating lung cancer as described in [1], whereinthe lung cancer is small cell lung cancer or squamous cell lungcarcinoma.

[4] The medicament for treating lung cancer as described in any of [1]to [3], wherein the amrubicin or a pharmaceutically acceptable saltthereof is amrubicin hydrochloride.

[5] The medicament for treating lung cancer as described in any of [1]to [4], wherein the other medicament for treating lung cancer isirinotecan, nogitecan, vinorelbine, vincristine, gemcitabine, 5-FU,paclitaxel, docetaxel or ZD 1839.

[6] The medicament for treating lung cancer as described in any of [1]to [4], wherein the other medicament for treating lung cancer isirinotecan, vinorelbine, gemcitabine, paclitaxel, docetaxel or ZD 1839.

[7] The medicament for treating lung cancer as described in any of [1]to [6], which is administered simultaneously with, separately from orsequentially with the other medicament for treating lung cancer.

[8] The medicament for treating lung cancer as described in any of [1]to [7], for a patient carrying a lung cancer to which the othermedicament for treating lung cancer has been administered or is plannedto be administered.

[9] The medicament for treating lung cancer as described in any of [1]to [8], which is packaged such that about 60 to about 150 mg/m² ofamrubicin or a pharmaceutically acceptable salt thereof is administeredin a single dose or in 2 to 5 divided doses.

[10] The medicament for treating lung cancer as described in [9], whichis packaged such that about 80 to about 130 mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered in a singledose.

[11] The medicament for treating lung cancer as described in [9], whichis packaged such that about 110 to about 130 mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered in a singledose.

[12] The medicament for treating lung cancer as described in [9], whichis packaged such that about 25 to about 50 Mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered once a day for3 days.

[13] The medicament for treating lung cancer as described in [9], whichis packaged such that about 30 to about 45 mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered once a day for3 days.

[14] The medicament for treating lung cancer as described in [9], whichis packaged such that about 35 to about 45 mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered once a day for3 days.

[15] The medicament for treating lung cancer as described in any of [12]to [14], wherein amrubicin or a pharmaceutically acceptable salt thereofis administered for continuous 3 days.

[16] The medicament for treating lung cancer as described in any of [1]to [15], wherein the other medicament for treating lung cancer isadministered in a combined use of about 0.4 times to about 1.0 time ofthe maximum tolerated dose or 0.4 times to about 1.0 time of the maximumadministered dose.

[17] The medicament for treating lung cancer as described in any of [1]to [16], for a patient having a lung cancer, wherein the patient is onehaving failed to continue receiving the treatment with the othermedicament for treating lung cancer because of side effects, and whereinthe patient is receiving administration of said other medicament in anamount that will cause reduced side effect.

[18] A use of amrubicin or a pharmaceutically acceptable salt thereoffor the manufacture of a medicament for treating lung cancer for a usein combination with another medicament for treating lung cancer.

[19] A method for treating a lung cancer which comprises administeringamrubicin or a pharmaceutically acceptable salt thereof and anothermedicament for treating lung cancer.

The medicament for treating lung cancer of the invention is a medicamentfor treating lung cancer comprising amrubicin or a pharmaceuticallyacceptable salt thereof as an active ingredient and is used incombination with another medicament for treating lung cancer.

Amrubicin or pharmaceutically acceptable salts thereof can be prepared,for example, according to J. Org. Chem., 52, 4477-4485 (1987). Thepharmaceutically acceptable salts of amrubicin include acid additionsalts and base addition salts. The acid addition salts include, forexample, inorganic acid salts such as hydrochloride, hydrobromide,sulfate, hydroiodide, nitrate, phosphate and the like; and organic acidsalts such as citrate, oxalate, acetate, formate, propionate, benzoate,trifluoroacetate, fumarate, maleate, tartrate, aspartate, glutamate,methanesulfonate, benzenesulfonate, camphorsulfonate and the like. Thebase addition salts include, for example, inorganic base addition saltssuch as sodium salt, potassium salt, calcium salt, magnesium salt,ammonium salt and the like; and organic base addition salts such astriethylammonium salt, triethanolammonium salt, pyridinium salt,diisopropylammonium salt and the like. Preferred pharmaceuticallyacceptable salts include hydrochloride and the like.

The maximum tolerated dose of amrubicin or pharmaceutically acceptablesalts thereof is, referring to amrubicin hydrochloride, 25 mg/kg (75mg/m²) for mice and 130 mg/m² for humans in once a day single dose, and50 mg/m² per day for administration in 3 continuous days.

Another medicament for treating lung cancer is any medicament fortreating lung cancer except amrubicin and pharmaceutically acceptablesalts, for example, it includes DNA topoisomerase I inhibitors (e.g.irinotecan, nogitecan), tublin polymerization inhibitors (e.g.vinorelbine, vincristine), antimetabolites (e.g. gemcitabine, 5-FU),tublin depolymerization inhibitors (e.g. paclitaxel, docetaxel) andtyrosine kinase inhibitors (e.g. ZD 1839).

Preferable is described below, for example, for the other medicament fortreating lung cancer.

-   Irinotecan: JP 60-19790A, U.S. Pat. No. 4,604,463, J. Clin. Oncol.,    11, 909(1993)-   Nogitecan: EP 0321122B1-   Vinorelbine: JP 55-31096A, U.S. Pat. No. 4,307,100, Cancer Letters,    27, 285 (1985)-   Vincristine-   Gemcitabine: U.S. Pat. No. 4,808,614, Cancer Treat. Rev., 19,    45-55 (1993) 5-FU (5-Fluorourasil)-   Paclitaxel: J. Am. Chem. Soc., 93, 2325-2327 (1971)-   Docetaxel: J. Natl. Cancer Inst., 83, 288-291 (1991) ZD 1839    (4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline;    generic name Gefitinib; Product name IRESSA): Drugs 2000: 60 Suppl.    1, 33-40    The maximum tolerated dose of irinotecan is 120 mg/kg for mice and    the maximum administered dose for humans is, for example, 150 mg/m²    at intervals of a week and 350 mg/m² at intervals of 3 weeks. The    maximum tolerated dose of vinorelbine is 16 mg/kg for mice and the    maximum administered dose for humans is 25 mg/m². The maximum    tolerated dose of gemcitabine is 300 mg/kg/dose for mice and the    maximum administered dose for humans is 1000 mg/m². The maximum    tolerated dose of paclitaxel is 12.5 mg/kg/dose for mice and the    maximum administered dose for humans is 210 mg/m² The maximum    tolerated dose of docetaxel is 30 mg/kg/dose for mice and the    maximum administered dose for humans is 70 mg/m². The maximum    tolerated dose of ZD 1839 is 200 mg/kg for mice and 700 mg/day for    humans, and the maximum administered dose for humans is about 500    mg/day.

The lung cancer includes, for example, small cell lung cancer, lungadenocarcinoma, squamous cell lung carcinoma, large cell lung carcinoma,carcinoids, adenoid cystic carcinoma, mucoepidermoid carcinoma,malignant mixed tumor and the like. Among them, examples in which themedicament for treating lung cancer of the invention exhibit preferredeffect include small cell lung cancer, lung adenocarcinoma, squamouscell lung carcinoma, large cell lung carcinoma and the like, andparticularly preferred one is small cell lung cancer and squamous celllung carcinoma.

From results of Examples 1 to 3 with mice, the following facts arefound:

(1) In a combined use of 0.5 times (12.5 mg/kg) of the maximum tolerateddose of amrubicin hydrochloride and 0.5 times of the maximum tolerateddose of the other medicament for treating lung cancer, an anticancereffect was observed at a similar level as compared with those found ingroups in which the maximum tolerated dose of each of respective agentswas administered alone. On the other hand, their side effects weresignificantly decreased.

(2) In a combined use of 0.8 times or 1.0 time (20 or 25 mg/kg) of themaximum tolerated dose of amrubicin hydrochloride and 0.8 times or 1.0time of the maximum tolerated dose of the other medicament for treatinglung cancer, a stronger anticancer effect was observed as compared withthose found in groups to which the maximum tolerated dose of each ofrespective agents was administered alone. On the other hand, their sideeffects were not increased but at a similar level.

As described above, by the combined use of about 0.5 to about 1 time ofthe maximum tolerated dose of amrubicin or a pharmaceutically acceptablesalt thereof and about 0.5 to about 1 time of the maximum tolerated doseof the other medicament for treating lung cancer, anticancer effect canbe obtained safely and sufficiently without increasing side effects ofsaid medicament for treating lung cancer and amrubicin orpharmaceutically acceptable salts thereof or sometimes decreasing theside effects. For example, when the side effects of said medicament fortreating lung cancer or amrubicin are taken up as a problem, a lowerdose within the range of about 0.5 to about 1 time of the maximumtolerated dose of amrubicin or a pharmaceutically acceptable saltthereof and about 0.5 to about 1 time of the maximum tolerated dose ofthe other medicament for treating, lung cancer can be applied; on theother hand, when the side effects of said medicament for treating lungcancer or amrubicin are not taken up as a problem, a higher dose withina range of about 0.5 to about 1 time of the maximum tolerated dose ofamrubicin or a pharmaceutically acceptable salt thereof and about 0.5 toabout 1 time of the maximum tolerated dose of said medicament fortreating lung cancer can be used for safely obtaining the maximumanticancer effect.

In the treatment of human lung cancers, while the amount may be suitablyvaried depending on conditions, age, body weight and so on of thepatient, for example, about 60 to about 150 mg/m² of amrubicin or apharmaceutically acceptable salt thereof and 0.4 to 1.0 time of themaximum tolerated dose (or maximum administered dose) of the othermedicament for treating lung cancer can be used in combination.Amrubicin or a pharmaceutically acceptable salt thereof can beadministered, for example, in an amount of about 60 to about 150 mg/m²in a single dose or in 2 to 5 divided doses. Preferred schedule foradministration of amrubicin or a pharmaceutically acceptable saltthereof includes, for example, a single administration, once dailyadministration for 3 days and the like, and includes most preferablyonce daily administration over 3 continuous days. The dose for a singleadministration includes, for example, a range of about 80 to about 130mg/m², and includes preferably about 110 mg/m² to about 130 mg/m² andmore preferably about 120 mg/m². The dose per day for administrationover 3 continuous days includes, for example, a range of about 25 toabout 50 mg/m², preferably a range of about 30 to about 45 mg/m², morepreferably a range of about 35 to about 45 mg/m², and most preferablyabout 40 mg/m², about 45 mg/m² and the like.

The amount of administration of the other medicament for treating lungcancer to be administered in combination includes, for example, a rangeof about 0.4 to about 1.0 time of the maximum tolerated dose or maximumadministered dose in a single dose, preferably about 0.5 to about 0.9times of the maximum tolerated dose or maximum administered dose in asingle dose. For example, it is about 75 to about 135 mg/m² foririnotecan, about 12.5 to about 22.5 mg/m² for vinorelbine, about 500 toabout 900 mg/m² for gemcitabine, about 105 to about 190 mg/m² forpaclitaxel, about 35 to about 63 mg/m² for docetaxel and about 350 toabout 630 mg/day for ZD 1839. With regard to ZD 1839, it is known that arather lower dosage gives effect, thus it is possible to provide theeffect in combination even in about 125 to about 630 mg/day. Inaddition, the other medicament for treating lung cancer can beadministered in several divided doses, within a day or over severaldays.

In patients carrying a lung cancer treated with the other medicament fortreating lung cancer, when it has been judged that the treatment can notbe continued because of side effects of said medicament, a treatmentwith decreased side effects of said medicament for treating lung cancercan be continued by an administration of said medicament for treatinglung cancer in a dose with reduced side effects and additionaladministration of amrubicin or a pharmaceutically acceptable saltthereof. The dose of said medicament for treating lung cancer forreduction of side effects includes, for example, a range of about 0.4 toabout 0.8 times of the maximum tolerated dose (or maximum administereddose) and preferably includes a range of about 0.4 to about 0.6 times ofthe maximum tolerated dose (or maximum administered dose).

In the medicament for treating lung cancer of the invention, amrubicinor a pharmaceutically acceptable salt thereof is administeredsimultaneously with, separately from or sequentially with the othermedicament for treating lung cancer. When it is administered separatelyor sequentially, amrubicin or a pharmaceutically acceptable salt thereofmay be administered before or be administered after the other medicamentfor treating lung cancer. The interval of both administration cansuitably be set and may be, for example, 1 to several hours, ten toseveral tens hours, 1 to several days, 1 week and the like. For example,it is preferred, in view of patient's convenience such as visitinghospital or the like, that amrubicin or a pharmaceutically acceptablesalt thereof and the other medicament for treating lung cancer areadministered on the same day.

While the administration of the medicament for treating lung cancer ofthe invention suitably varies depending on conditions, age and bodyweight of patient, form for administration, amount for administration ofthe other medicament for treating lung cancer to be administered incombination, frequency of administration and the like, it is preferredthat the both administrations are repeated after the administration ofthe above amrubicin or a pharmaceutically acceptable salt thereof andthat of the other medicament for treating lung cancer at an interval ofabout 7 days to about 60 days. Most preferably, repetition is made at aninterval of about 2 weeks to about 4 weeks and further preferably at aninterval of about 3 weeks.

Amrubicin or a pharmaceutically acceptable salt thereof can usually beadministered parenterally (for example, intravenous, intraarterial,subcutaneous or intramuscular injection; intravesically,intraperitoneally, intrapleurally, topically, rectally, percutaneously,nasally and so on). Preferred route includes intravenous injection. Inaddition, oral administration is also possible, and forms for oraladministration include tablets, capsules, pills, granules, powders,solutions, syrups, suspensions and the like.

The other medicament for treating lung cancer can usually beadministered parenterally (for example, intravenous, intraarterial,subcutaneous or intramuscular injection; intravesically,intraperitoneally, intrapleurally, topically, rectally, percutaneously,nasally and so on). Preferred route includes intravenous injection. Inaddition, oral administration is also possible, and forms for oraladministration include tablets, capsules, pills, granules, powders,solutions, syrups, suspensions and the like.

In the medicament for treating lung cancer of the invention, further theother anticancer agent, irradiation therapy, surgical measures and thelike can further be combined. Additionally, it can be in the form of akit for combined medicament for treating lung cancer comprising (a) afirst composition comprising amrubicin or a pharmaceutically acceptablesalt thereof as an active ingredient and (b) a second compositioncomprising another medicament for treating lung cancer as an activeingredient.

EXAMPLES

The invention is described below in more detail with reference toExamples, which do not limit the invention.

Example 1

Antitumor Activity by a Combination of Amrubicin Hydrochloride andIrinotecan:

Human small cell lung cancer LX-1 cell strain was subcutaneouslytransplanted to nude mice (59 animals) at 6 weeks of age. After 11 daysfrom the tumor transplantation, 36 animals having a tumor volume ofabout 100 to 300 mm³ were allotted to 6 groups consisting of 6 animalsper group. On the same day, the animals received intravenousadministration, respectively, of cysteine buffer for “vehicle group”,the maximum tolerated dose of amrubicin hydrochloride (25 mg/kg) for “anamrubicin hydrochloride alone administration group”, the maximumtolerated dose of irinotecan (120 mg/kg) for “irinotecan aloneadministration group”, 0.5 times of the maximum tolerated dose ofamrubicin hydrochloride and 0.5 times of the maximum tolerated dose ofirinotecan for “combined administration group (0.5×MTD)”, 0.8 times ofthe maximum tolerated dose of amrubicin hydrochloride and 0.8 times ofthe maximum tolerated dose of irinotecan for “combined administrationgroup (0.8×MTD)”, the maximum tolerated dose of amrubicin hydrochlorideand the maximum tolerated dose of irinotecan for “combinedadministration group (1×MTD)”. Thereafter, tumor volume and body weightof the mice were measured for 23 days.

Amrubicin hydrochloride was dissolved in a cysteine buffer (containing0.4 mg/ml L-cysteine hydrochloride monohydrate and 6.25 mg/ml lactose)to give a solution of 2.5 mg/ml, which was diluted with a cysteinebuffer to give solutions of 2.0 and 1.25 mg/ml. Each 10 ml/kg aliquot ofthe solutions was administered as the maximum tolerated dose, the 0.8times dose or the 0.5 times dose of amrubicin hydrochloride.

Irinotecan was administered by giving 120, 96 or 60 mg/kg aliquot ofTopotecin Injection (containing 20 mg/ml), purchased from DaiichiPharmaceutical Co., Ltd., as the maximum tolerated dose, the 0.8 timesdose or the 0.5 times dose of irinotecan.

FIGS. 1 and 2 show changes in tumor volume and body weight for thecombined administration (0.5×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for theirinotecan alone administration group.

FIGS. 3 and 4 show changes in tumor volume and body weight for thecombined administration (0.8×MTD) group together with data for theamrubicin hydrochloride independent administration group and those forthe irinotecan independent administration group.

FIGS. 5 and 6 show changes in tumor volume and body weight for thecombined administration (1×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for theirinotecan alone administration group.

TABLE 1 shows the minimum T/C % of tumor growth rate in respectivegroups. The minimum T/C % was calculated as follows:

Minimum T/C %: the minimum value of ratio (%) of tumor growth rate^(*))for respective administration groups to tumor growth rate^(*)) for thevehicle administration group within the period for measurement.^(*))Tumor growth rate: a ratio of an average value of tumor volume fora group of 6 animals at respective point in time of measurement to anaverage value of tumor volume for a group of 6 animals at respectivepoint in time of drug administration.TABLE 1 Minimum T/C(%) Administration Average ± SD Amrubicinhydrochloride 25 mg/kg 40.79 ± 5.92 Irinotecan 120 mg/kg 45.61 ± 12.94Amrubicin hydrochloride 25 mg/kg + Irinotecan 24.24 ± 5.48 120 mg/kgAmrubicin hydrochloride 20 mg/kg + Irinotecan 28.17 ± 3.85 96 mg/kgAmrubicin hydrochloride 12.5 mg/kg + Irinotecan 45.05 ± 4.74 60 mg/kg1. Results in the Combined Administration (0.5×MTD) Group:

As shown in FIG. 1, the antitumor effect in the combined administrationwas a similar effect as compared with those in the maximum tolerateddose administration groups for respective single agents. Thus, theminimum T/C % was 40.79% in the amrubicin hydrochloride aloneadministration group, 45.61% in the irinotecan alone administrationgroup and 45.05% in the combined administration (0.5×MTD) group.

The side effects as evaluated by loss in the body weight of animals, asshown in FIG. 2, was at a similar degree to those in the irinotecanalone administration group as compared with the single administrationgroups for respective single agents.

2. Results in the Combined Administration (0.8×MTD) Group:

As shown in FIG. 3, there was a reduction of tumor, and the antitumoreffect in the combined administration was a stronger antitumor effect ascompared with those in the single administration groups for respectivesingle agents. Thus, the minimum T/C % was 40.79% in the amrubicinhydrochloride alone administration group, 45.61% in the irinotecan aloneadministration group and 28.17% in the combined administration (0.8×MTD)group.

The side effects as evaluated by loss in the body weight of animals, asshown in FIG. 4, was at a similar degree to those in the amrubicinhydrochloride alone administration group.

3. Results in the Combined Administration (1×MTD) Group:

As shown in FIG. 5, there was a reduction of tumor as in the case of 0.8times administration, and the antitumor effect in the combinedadministration was a stronger antitumor effect as compared with those inthe single administration groups for respective single agents. Thus, theminimum T/C % was 40.79% in the amrubicin hydrochloride aloneadministration group, 45.61% in the irinotecan alone administrationgroup and 24.24% in the combined administration (1×MTD) group.

For the side effects as evaluated by loss in the body weight of animals,as shown in FIG. 6, there was a transitory loss in the body weight byabout 3 g, but the weight recovered.

Example 2

Antitumor Activity by a Combination of Amrubicin Hydrochloride andVinorelbine:

Antitumor activity by a combination of amrubicin hydrochloride andvinorelbine was tested in the same way as Example 1, except that humansquamous cell lung carcinoma QC-56 cell strain was used in place ofhuman small cell lung cancer LX-1 cell strain and that the maximumtolerated dose of vinorelbine was set at 16 mg/kg. Vinorelbine wasadministered by giving 16, 12.8 or 8 ml/kg aliquot of NavelbineInjection (containing 10 mg/ml), purchased from Kyowa Hakko Kogyo Co.,Ltd., as the maximum tolerated dose, the 0.8 times dose or the 0.5 timesdose of vinorelbine. In addition, nude mice (100 animals) at 5 weeks ofage were used.

FIGS. 7 and 8 show changes in tumor volume and body weight for thecombined administration (0.5×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for thevinorelbine alone administration group.

FIGS. 9 and 10 show changes in tumor volume and body weight for thecombined administration (0.8×MTD) group together with data for theamrubicin hydrochloride independent administration group and those forthe vinorelbine independent administration group.

FIGS. 11 and 12 show changes in tumor volume and body weight for thecombined administration (1×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for thevinorelbine alone administration group.

TABLE 2 shows the minimum T/C % of tumor growth rate in respectivegroups. TABLE 2 Minimum T/C(%) Administration Average ± SD Amrubicinhydrochloride 25 mg/kg 37.73 ± 7.56 Vinorelbine 16 mg/kg 38.24 ± 12.87Amrubicin hydrochloride 25 mg/kg + Vinorelbine 27.15 ± 8.14 16 mg/kgAmrubicin hydrochloride 20 mg/kg + Vinorelbine 31.10 ± 13.48 12.8 mg/kgAmrubicin hydrochloride 12.5 mg/kg + Vinorelbine 42.43 ± 20.08 8 mg/kg1. Results in the Combined Administration (0.5×MTD) Group:

As shown in FIG. 7, the antitumor effect in the combined administrationwas a similar effect as compared with those in the maximum tolerateddose administration groups for respective single agents. Thus, theminimum T/C % was 37.73% in the amrubicin hydrochloride aloneadministration group, 38.24% in the vinorelbine alone administrationgroup and 42.43% in the combined administration (0.5×MTD) group.

For the side effects as evaluated by loss in the body weight of animals,as shown in FIG. 8, the increase of the side effect was not observedbecause of rare loss in the body weight compared with the singleadministration groups for respective single agents.

2. Results in the Combined Administration (0.8×MTD) Group:

As shown in FIG. 9, there was a reduction of tumor, and the antitumoreffect in the combined administration was a stronger antitumor effect ascompared with those in the single administration groups for respectivesingle agents. Thus, the minimum T/C % was 37.73% in the amrubicinhydrochloride alone administration group, 38.24% in the vinorelbinealone administration group and 31.10% in the combined administration(0.8×MTD) group.

The side effects as evaluated by loss in the body weight of animals, asshown in FIG. 10, was at a similar degree to those in the singleadministration groups for respective single agents.

3. Results in the Combined Administration (1×MTD) Group:

As shown in FIG. 11, there was a reduction of tumor as in the case of0.8 times administration, and the antitumor effect in the combinedadministration was a stronger antitumor effect as compared with those inthe single administration groups for respective single agents. Thus, theminimum T/C % was 37.73% in the amrubicin hydrochloride aloneadministration group, 38.24% in the vinorelbine alone administrationgroup and 27.15% in the combined administration (1×MTD) group.

For the side effects as evaluated by loss in the body weight of animals,as shown in FIG. 12, there was a transitory loss in the bodyweight byabout 1.4 g, but the weight recovered.

Example 3

Antitumor Activity by a Combination of Amrubicin Hydrochloride andGemcitabine:

Antitumor activity by a combination of amrubicin hydrochloride andgemcitabine was tested in the same way as Example 2, except that themaximum tolerated dose of gemcitabine was set at 300 mg/kg/day and theadministration was intraperitoneally once per week in two cycles.Gemcitabine was administered by giving 300, 240 or 150 ml/kg aliquot ofGemzar Injection (containing 200 mg/bial), purchased from Eli LillyJapan K.K., as the maximum tolerated dose, the 0.8 times dose or the 0.5times dose of gemcitabine. In addition, nude mice (80 animals) at 5weeks of age were used.

FIGS. 13 and 14 show changes in tumor volume and body weight for thecombined administration (0.5×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for thegemcitabine alone administration group.

FIGS. 15 and 16 show changes in tumor volume and body weight for thecombined administration (0.8×MTD) group together with data for theamrubicin hydrochloride independent administration group and those forthe gemcitabine independent administration group.

FIGS. 17 and 18 show changes in tumor volume and body weight for thecombined administration (1×MTD) group together with data for theamrubicin hydrochloride alone administration group and those for thegemcitabine alone administration group.

TABLE 3 shows the minimum T/C % of tumor growth rate in respectivegroups. TABLE 3 Minimum T/C(%) Administration Average ± SD Amrubicinhydrochloride 25 mg/kg 51.06 ± 16.56 Gemcitabine 300 mg/kg i.p. 2q7d69.47 ± 26.06 Amrubicin hydrochloride 25 mg/kg + Gemcitabine 41.33 ±15.12 300 mg/kg Amrubicin hydrochloride 20 mg/kg + Gemcitabine 34.27 ±14.05 240 mg/kg Amrubicin hydrochloride 12.5 mg/kg + Gemcitabine 56.67 ±18.87 150 mg/kg1. Results in the Combined Administration (0.5×MTD) Group:

As shown in FIG. 13, the antitumor effect in the combined administrationwas a similar effect as compared with those in the maximum tolerateddose administration groups for respective single agents. Thus, theminimum T/C % was 51.06% in the amrubicin hydrochloride aloneadministration group, 69.47% in the gemcitabine alone administrationgroup and 56.67% in the combined administration (0.5×MTD) group.

For the side effects as evaluated by loss in the body weight of animals,as shown in FIG. 14, a remarkable reducing effect of the side effects,possibly caused by halving the dose of gemcitabine, was observed ascompared with the single administration groups for respective singleagents.

2. Results in the Combined Administration (0.8×MTD) Group:

As shown in FIG. 15, there was a reduction of tumor, and the antitumoreffect in the combined administration was a stronger antitumor effect ascompared with those in the single administration groups for respectivesingle agents. Thus, the minimum T/C % was 51.06% in the amrubicinhydrochloride alone administration group, 69.47% in the gemcitabinealone administration group and 34.27% in the combined administration(0.8×MTD) group.

The side effects as evaluated by loss in the body weight of animals, asshown in FIG. 16, was at a similar degree to those in the amrubicinhydrochloride alone administration group.

3. Results in the Combined Administration (1×MTD) Group:

As shown in FIG. 17, there was a reduction of tumor as in the case of0.8 times administration, and the antitumor effect in the combinedadministration was a stronger antitumor effect as compared with those inthe single administration groups for respective single agents. Thus, theminimum T/C % was 51.06% in the amrubicin hydrochloride aloneadministration group, 69.47% in the gemcitabine alone administrationgroup and 41.33% in the combined administration (1×MTD) group.

The side effects as evaluated by loss in the body weight of animals, asshown in FIG. 18, was at a similar degree to those in the gemcitabinealone administration group.

As described in the above Examples 1 to 3, side effects were reduced andremarkable therapeutic effects were observed in the combinedadministration of amrubicin hydrochloride with irinotecan, vinorelbineand gemcitabine. In addition, as it can be understood from FIGS. 1 to18, the effects by the combined use appear with particular significanceduring 2 weeks from the administration and the effects almost disappearafter 3 weeks. Consequently, it is preferred that the administration berepeated again after about 2 weeks to about 4 weeks, and particularly,re-administration is carried out after about 3 weeks and continuedthereafter.

With regard to paclitaxel (the maximum tolerated dose, 12.5 mg/kg/dose),the effects by the combined use with amrubicin hydrochloride can beconfirmed similar to Example 1. Further, with regard to ZD 1839 (themaximum tolerated dose, 200 mg/kg), the effects by the combined use withamrubicin hydrochloride can be confirmed by carrying out the similartest to Example 1 provided that human squamous cell carcinoma A431 cellstrain was used.

Example 4

In Vitro Combination Effects of Amrubicinol Hydrochloride withIrinotecan:

The in vitro combination effects of amrubicinol hydrochloride withirinotecan hydrochloride were examined by using human non-small celllung cancer cell.

Human non-small cell lung cancer cell strain A549 was obtained from ATCC(American Type Culture Collection). In a medium comprising D-MEM(Dulbecco's Modified Eagle Medium) and 10% bovine fetal serum (FCS),human non-small cell lung cancer cell strain A549 was subcultured. Theculture was carried out in a incubator with 5% CO₂ at 37° C. The samemedium was also used in the following experiments.

Test agents were prepared as below.

Irinotecan hydrochloride was obtained from Daiichi Pharmaceutical Co.,Ltd. and provided after 2 fold serial dilutions with medium in use.

Amrubicinol was prepared by a method described in a literature (Ishizumiet al., J. Org. Chem., 52, 4477-4485 (1987)). Amrubicinol hydrochloridemaintained in a deep freezer (−80° C.) was measured each of about 1 mg,kept in a freezer (−20° C.), and then dissolved in distilled water togive 1 mg/ml in use, which was used after sterilization by filtrationfollowed by 2 fold serial dilution with the medium.

Human non-small cell lung cancer cell strain A549 on subculture wassubjected to a trypsin treatment, suspended in the medium and seeded ona 96-well plate. The seeding density was set to be 5×10² cells/0.1ml/well. After seeding, cells were cultured overnight in an incubatorwith 5% CO₂ at 37° C. (Day 0).

To the group for evaluating a single agent, were added 0.05 ml/well of adiluted solution of a test agent and 0.05 ml/well of the medium; and tothe group for evaluating the combination effect, were added 0.05 ml/wellof a diluted solution of amrubicinol hydrochloride and a dilutedsolution of 0.05 ml/well of irinotecan hydrochloride (Day 1). Theexperiments were conducted with n=3 for the agent-treated group, andwith n=6 for the untreated (control) group.

Until Day 4, culture was conducted in an incubator with 5% CO₂ at 37° C.

On Day 4, 0.02 ml of WST solution (a solution containing 1.3 mg/ml WST-1(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,sodium salt) and 0.14 mg/ml 1-Methoxy PMS (1-Methoxy-5-methylphenaziniummethyl sulfate) in a phosphate buffer solution (PBS)) was added to eachof the wells. Thereafter, culture was conducted in an incubator with 5%CO₂ at 37° C. for 2-4 hours. Absorbance of the culture was then measuredwith MICROPLATE READER Model 3550-UV (BioRad), and living cell numberwas measured.

Growth rate f was determined by the following formula:f=(mean value of absorbance at each of the agent concentrations)/(meanvalue of absorbance at the agent concentration of 0 mg/ml)

-   -   Absorbance: A₄₂₀−A₆₃₀        log((1/f)−1) was plotted versus log(agent concentration), and        then a regression line was drawn in accordance with a least        square method to find the slope m and X intercept. Exp(X        intercept value) represents IC₅₀ value (concentration of the        agent required for 50% inhibition of the cell growth). While the        ratio of the concentrations of amrubicinol hydrochloride and        irinotecan hydrochloride is 1:25, m and IC₅₀ value were obtained        respectively. (Hereinafter, IC₅₀ value is represented as D_(m),        and amrubicinol hydrochloride is represented with the subscript        1 while irinotecan hydrochloride is represented with the        subscript 2. e.g.: D_(m1))

Concentration D of amrubicinol hydrochloride and irinotecanhydrochloride required for achieving a particular inhibition rate ofgrowth (fa) was determined by the formula {circle over (1)}. Theconcentration for the single agent was defined as Df₁ and Df₂, while theconcentration when used in combination was defined as Dx₁ and Dx₂, andthus a Combination Index (CI) was calculated by the formula {circle over(2)} (M. Pegram et al., Oncogene, 18, 2241-2251 (1999)).D=D _(m) ×{fa/(1−fa)}^(1/m)  {circle over (1)}CI=Dx ₁ /Df ₁ +Dx ₂ /Df ₂+(Dx ₁ ×Dx ₂)/(Df ₁ ×Df ₂)  {circle over (2)}Evaluation is made as an additive effect at CI=1, as a synergisticeffect at CI<1, and as an antagonistic effect at CI>1.

FIG. 19 shows in vitro combination effects of amrubicinol hydrochloridewith irinotecan hydrochloride, in which a horizontal axis represents aninhibition rate of growth fa and a vertical axis represents CombinationIndex (CI). As shown in FIG. 19, the Combination Index (CI) indicated alower value of 1 at the range of fa=0.1 to 0.9. Thus, in the combinationof amrubicinol hydrochloride with irinotecan hydrochloride, a largesynergistic effect was revealed according to the determination of thecombination effect by CI.

Example 5

Fluctuation Analysis for Expression of Carbonyl Reductase Gene in Normaland Tumor Tissues:

DNA chip analysis was conducted using total RNAs prepared with 69samples from human lung normal tissue, and 44 samples from lungadenocarcinoma, 32 samples from squamous cell lung carcinoma, 5 samplesfrom large cell lung carcinoma and 18 samples from leukemia cell. TheDNA chip analysis was carried out with Gene Chip Human Genome U95A, B,C, D and E from Affymetrix. Specifically, the analysis was performedwith a procedure consisting of (1) preparation of a cDNA from a totalRNA, (2) preparation of a labeled cRNA from said cDNA, (3) fragmentationof the labeled cRNA, (4) hybridization of the fragmented cRNA with aprobe array, (5) staining of the probe array, (6) scanning of the probearray and (7) analysis of gene expression.

(1) Preparation of a cDNA from a Total RNA:

Each of 11 μl of mixed solutions containing 10 μg of each of total RNAsprepared with 69 samples from human lung normal tissue, and 44 samplesfrom lung adenocarcinoma, 32 samples from squamous cell lung carcinoma,5 samples from large cell lung carcinoma and 18 samples from leukemiacell and 100 pmols of T7−(dT)24 primer (manufactured by Amersham) washeated at 70° C. for 10 minutes and cooled on ice. After cooling, 4 μlof 5× First Strand cDNA Buffer contained in SuperScript Choice Systemfor cDNA Synthesis (manufactured by Gibco-BRL), 2 μl of 0.1 M DTT(dithiothreitol) contained in said kit and 1 μl of 10 mM dNTP Mix wereadded and the mixture was heated at 42° C. for 2 minutes. Further, 2 μl(400 U) of SuperScript II RT contained in said kit was added. Themixture was heated at 42° C. for 1 hour and cooled on ice. Aftercooling, 91 μl of DEPC treated water (manufactured by Nacalai Tesque,Inc.), 30 μl of 5× Second Strand Reaction Buffer contained in said kit,3 μl of 10 mM dNTP Mix, 1 μl (10 U) of E. coli DNA Ligase contained insaid kit, 4 μl (40 U) of E. coli DNA Polymerase I contained in said kitand 1 μl (2 U) of E. coli RNAaseH contained in said kit were added andreacted at 16° C. for 2 hours. Then, after adding 2 μl (10 U) of T4 DNAPolymerase contained in said kit and reacting at 16° C. for 5 minutes,10 μl of 0.5 M EDTA was added. Then, 162 μl of phenol/chloroform/isoamylalcohol solution (manufactured by Nippongene) was added and mixed. Themixed solution was transferred to Phase Lock Gel Light (manufactured byEppendorf), which was previously centrifuged at room temperature and14,000 rpm for 30 seconds, centrifuged at room temperature and 14,000rpm for 2 minutes and 145 μl of aqueous layer was transferred to anEppendorf tube. To the obtained solution were added 72.5 μl of 7.5 Mammonium acetate solution and 362.5 μl of ethanol, and after mixing, themixture was centrifuged at 4° C. and 14,000 rpm for 20 minutes. Aftercentrifugation, the supernatant was discarded to give a DNA pelletcontaining the prepared cDNA. Then, 0.5 ml of 80% ethanol was added tosaid pellet and the mixture was centrifuged at 4° C. and 14,000 rpm for5 minutes. The supernatant was discarded. After repeating again the sametreatment, the pellet was dried and dissolved in 12 μl of DEPC treatedwater.

By the above procedure, cDNAs were obtained from total RNAs derived from69 samples from human lung normal tissue, and 44 samples from lungadenocarcinoma, 32 samples from squamous cell lung carcinoma, 5 samplesfrom large cell lung carcinoma and 18 samples from leukemia cell.

(2) Preparation of a Labeled cRNA from Said cDNA:

To 5 μl of each of the cDNA solutions prepared in the above (1) weremixed 17 μl of DEPC treated water, 4 μl of 10×HY Reaction Buffercontained in BioArray High Yield RNA Transcript Labeling Kit(manufactured by ENZO), 4 μl of 10× Biotin Labeled Ribonucleotidescontained in said kit, 4 μl of 10×DTT contained in said kit, 4 μl of 10×RNase Inhibitor Mix contained in said kit and 2 μl of 20×T7 RNAPolymerase contained in said kit, and reacted at 37° C. for 5 hours.After the reaction, 60 μl of DEPC treated water was added to thereaction solution and the prepared labeled cRNAs were purified withRNeasy Mini Kit according to the attached protocol.

(3) Fragmentation of the Labeled cRNA:

To a solution containing 20 μg of each of labeled cRNAs purified in theabove (3) were added 8 μl of 5× Fragmentation Buffer (200 mMtris-acetate, pH 8.1 (manufactured by Sigma), 500 mM potassium acetate(manufactured by Sigma) and 150 mM magnesium acetate (manufactured bySigma)). After heating 40 μl of the obtained reaction solution at 94° C.for 35 minutes, the solution was placed in ice. This allowedfragmentation of the labeled cRNAs.

(4) Hybridization of the Fragmented cRNA with a Probe Array:

To 40 μl of each of the fragmented cRNAs obtained in the above (3) weremixed 4 μl of 5 nM Control Oligo B2 (manufactured by Amersham), 4 μl of100× Control cRNA Cocktail, 40 μg of Herring sperm DNA (manufactured byPromenga), 200 μg of Acetylated BSA (manufactured by Gibco-BRL), 200 μlof 2×MES Hybridization Buffer (200 mM MES, 2 M [Na⁺], 40 mM EDTA, 0.02%Tween 20 (manufactured by Pierce), pH 6.5-6.7) and 144 μl of DEPCtreated water to give 400 μl hybridized cocktail. Each of the obtainedhybridized cocktails was heated at 99° C. for 5 minutes, andadditionally at 45° C. for 5 minutes. After heating, the cocktail wascentrifuged at room temperature and 14,000 rpm for 5 minutes to give asupernatant of the hybridized cocktail.

On the other hand, after rotating Human genome U95 probe array(manufactured by Affymetrix) filled with 1×MES hybridization buffer in ahybridization oven at 45° C. and 60 rpm for 10 minutes, 1×MEShybridization buffer was removed to give a probe array. To the probearray was added 200 μl of the supernatant of the hybridized cocktailobtained above, and the mixture was rotated in a hybridization oven at45° C. and 60 rpm for 16 hours to give a probe array hybridized withfragmented cRNA.

(5) Staining of the Probe Array:

After collecting and removing the hybridized cocktail from each of thealready hybridized probe array obtained in the above (4), the productwas filled with Non-Stringent Wash Buffer (6×SSPE (prepared by diluting20×SSPE (manufactured by Nacalai Tesque)), 0.01% Tween 20 and 0.005%Antifoam 0-30 (manufactured by Sigma)). Then, the fragmented cRNA andhybridized probe array were placed in respective positions of GeneChipFluidics Station 400 (manufactured by Affymetrix) set with Non-StringentWash Buffer and Stringent Wash Buffer (100 mM MES, 0.1 M NaCl and 0.01%Tween 20). Then according to the staining protocol EuKGE-WS2, stainingwas carried out with a first staining solution (10 μg/ml StreptavidinPhycoerythrin (SAPE) (manufactured by Molecular Probe), 2 mg/mlAcetylated BSA, 100 mM MES, 1 M NaCl (manufactured by Ambion), 0.05%Tween 20 and 0.005% Antifoam 0-30) and a second staining solution (100μg/ml Goat IgG (manufactured by Sigma), 3 μg/ml BiotinylatedAnti-Streptavidin antibody (manufactured by Vector Laboratories), 2mg/ml Acetylated BSA, 100 mM MES, 1 M NaCl, 0.05% Tween 20 and 0.005%Antifoam 0-30).

(6) Scanning of the Probe Array and (7) Analysis of Gene Expression:

Each of probe arrays stained in the above (5) was subjected to HPGeneArray Scanner (manufactured by Affymetrix) to read the stainingpattern.

Expression of carbonyl reductase 1 gene on the probe array was analyzedwith GeneChip Workstation System (manufactured by Affymetrix) based onthe staining pattern. Then, normalization and comparative analysis ofgene expression were preformed according to the analysis protocol.

As the result, in human leukemia cells, it was found that the expressionfrequency of carbonyl reductase 1 was 11% (2 cases in 18 cases) andmedian of expression level was−39, indicating that the gene is hardlyexpressed. On the other hand, in human lung tissues, it was found thatthe expression frequency of carbonyl reductase 1 was 55% (24 cases in 44cases), 63% (20 cases in 32 cases), 40% (2 cases in 5 cases) and 32% (22cases in 69 cases) in adenocarcinoma, squamous cell carcinoma, largecell carcinoma and normal tissue, respectively, and expression level was51, 96, 34 and 22, respectively, indicating that the expression ofcarbonyl reductase 1 was enhanced in lung cancer tissues as comparedwith the lung normal tissue and, particularly, the expression levels inlung adenocarcinoma and squamous cell carcinoma were more than 2 timesand 4 times that in lung normal tissue, respectively.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an inhibitory effect of growth of small cell lung cancercell when 0.5 times of the maximum tolerated dose (MTD) of amrubicinhydrochloride and 0.5 times of the maximum tolerated dose of irinotecanare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes anirinotecan alone administration group and ▴ denotes a combinedadministration group.

FIG. 2 shows a reducing effect of the body weight as a side effect when0.5 times of the maximum tolerated dose of amrubicin hydrochloride and0.5 times of the maximum tolerated dose of irinotecan are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes an irinotecan aloneadministration group and ▴ denotes a combined administration group.

FIG. 3 shows an inhibitory effect of growth of small cell lung cancercell when 0.8 times of the maximum tolerated dose of amrubicinhydrochloride and 0.8 times of the maximum tolerated dose of irinotecanare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes anirinotecan alone administration group and ♦ denotes a combinedadministration group.

FIG. 4 shows a reducing effect of the body weight as a side effect when0.8 times of the maximum tolerated dose of amrubicin hydrochloride and0.8 times of the maximum tolerated dose of irinotecan are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes an irinotecan aloneadministration group and ♦ denotes a combined administration group.

FIG. 5 shows an inhibitory effect of growth of small cell lung cancercell when 1.0 time of the maximum tolerated dose of amrubicinhydrochloride and 1.0 time of the maximum tolerated dose of irinotecanare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes anirinotecan alone administration group and ● denotes a combinedadministration group.

FIG. 6 shows a reducing effect of the body weight as a side effect when1.0 time of the maximum tolerated dose of amrubicin hydrochloride and1.0 time of the maximum tolerated dose of irinotecan are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes an irinotecan aloneadministration group and ● denotes a combined administration group.

FIG. 7 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 0.5 times of the maximum tolerated dose of amrubicinhydrochloride and 0.5 times of the maximum tolerated dose of vinorelbineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes avinorelbine alone administration group and ▴ denotes a combinedadministration group.

FIG. 8 shows a reducing effect of the body weight as a side effect when0.5 times of the maximum tolerated dose of amrubicin hydrochloride and0.5 times of the maximum tolerated dose of vinorelbine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes a vinorelbine aloneadministration group and ▴ denotes a combined administration group.

FIG. 9 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 0.8 times of the maximum tolerated dose of amrubicinhydrochloride and 0.8 times of the maximum tolerated dose of vinorelbineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes avinorelbine alone administration group and ♦ denotes a combinedadministration group.

FIG. 10 shows a reducing effect of the body weight as a side effect when0.8 times of the maximum tolerated dose of amrubicin hydrochloride and0.8 times of the maximum tolerated dose of vinorelbine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes a vinorelbine aloneadministration group and ♦ denotes a combined administration group.

FIG. 11 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 1.0 time of the maximum tolerated dose of amrubicinhydrochloride and 1.0 time of the maximum tolerated dose of vinorelbineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes avinorelbine alone administration group and ● denotes a combinedadministration group.

FIG. 12 shows a reducing effect of the body weight as a side effect when1.0 time of the maximum tolerated dose of amrubicin hydrochloride and1.0 time of the maximum tolerated dose of vinorelbine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes a vinorelbine aloneadministration group and ● denotes a combined administration group.

FIG. 13 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 0.5 times of the maximum tolerated dose of amrubicinhydrochloride and 0.5 times of the maximum tolerated dose of gemcitabineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes agemcitabine alone administration group and ▴ denotes a combinedadministration group.

FIG. 14 shows a reducing effect of the body weight as a side effect when0.5 times of the maximum tolerated dose of amrubicin hydrochloride and0.5 times of the maximum tolerated dose of gemcitabine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes a gemcitabine aloneadministration group and ▴ denotes a combined administration group.

FIG. 15 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 0.8 times of the maximum tolerated dose of amrubicinhydrochloride and 0.8 times of the maximum tolerated dose of gemcitabineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes agemcitabine alone administration group and ♦ denotes a combinedadministration group.

FIG. 16 shows a reducing effect of the body weight as a side effect when0.8 times of the maximum tolerated dose of amrubicin hydrochloride and0.8 times of the maximum tolerated dose of gemcitabine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, ε denotes a gemcitabine aloneadministration group and ♦ denotes a combined administration group.

FIG. 17 shows an inhibitory effect of growth of squamous cell lungcarcinoma cell when 1.0 time of the maximum tolerated dose of amrubicinhydrochloride and 1.0 time of the maximum tolerated dose of gemcitabineare used in combination. Line denotes a vehicle group, □ denotes anamrubicin hydrochloride alone administration group, Δ denotes agemcitabine alone administration group and ● denotes a combinedadministration group.

FIG. 18 shows a reducing effect of the body weight as a side effect when1.0 time of the maximum tolerated dose of amrubicin hydrochloride and1.0 time of the maximum tolerated dose of gemcitabine are used incombination. Line denotes a vehicle group, □ denotes an amrubicinhydrochloride alone administration group, Δ denotes a gemcitabine aloneadministration group and ● denotes a combined administration group.

FIG. 19 shows in vitro effects by combination of amrubicinolhydrochloride and irinotecan. The CI values are plotted from 0.1 to 0.9by the fa value, which shows a growth inhibition rate, on a horizontalaxis and CI (combination index) value on a vertical axis.

INDUSTRIAL APPLICABILITY

According to the invention, medicament for treating lung cancer combinedamrubicin hydrochloride with another medicament for treating lung canceruseful in the treatment of a subject of lung cancer is provided. By thecombined use with the other medicament for treating lung cancer, theantitumor therapeutic effect of amrubicin hydrochloride can be improvedand cancer therapy with reduced side effects of said medicament fortreating lung cancer becomes possible.

1. A medicament for treating lung cancer, comprising amrubicin or apharmaceutically acceptable salt thereof as an active ingredient, for ause in combination with another medicament for treating lung cancer. 2.The medicament for treating lung cancer as described in claim 1, whereinthe lung cancer is small cell lung cancer, lung adenocarcinoma, squamouscell lung carcinoma or large cell lung carcinoma.
 3. The medicament fortreating lung cancer as described in claim 1, wherein the lung cancer issmall cell lung cancer or squamous cell lung carcinoma.
 4. Themedicament for treating lung cancer as described in any of claims 1 to3, wherein the amrubicin or a pharmaceutically acceptable salt thereofis amrubicin hydrochloride.
 5. The medicament for treating lung canceras described in claim 1, wherein the other medicament for treating lungcancer is irinotecan, nogitecan, vinorelbine, vincristine, gemcitabine,5-FU, paclitaxel, docetaxel or ZD
 1839. 6. The medicament for treatinglung cancer as described in claim 1, wherein the other medicament fortreating lung cancer is irinotecan, vinorelbine, gemcitabine,paclitaxel, docetaxel or ZD
 1839. 7. The medicament for treating lungcancer as described in claim 1, which is administered simultaneouslywith, separately from or sequentially with the other medicament fortreating lung cancer.
 8. The medicament for treating lung cancer asdescribed in claim 1, for a patient carrying a lung cancer to which theother medicament for treating lung cancer has been administered or isplanned to be administered.
 9. The medicament for treating lung canceras described in claim 1, which is packaged such that about 60 to about150 mg/m² of amrubicin or a pharmaceutically acceptable salt thereof isadministered in a single dose or in 2 to 5 divided doses.
 10. Themedicament for treating lung cancer as described in claim 9, which ispackaged such that about 80 to about 130 mg/m² of amrubicin or apharmaceutically acceptable salt thereof is administered in a singledose.
 11. The medicament for treating lung cancer as described in claim9, which is packaged such that about 110 to about 130 mg/m² of amrubicinor a pharmaceutically acceptable salt thereof is administered in asingle dose.
 12. The medicament for treating lung cancer as described inclaim 9, which is packaged such that about 25 to about 50 mg/m² ofamrubicin or a pharmaceutically acceptable salt thereof is administeredonce a day for 3 days.
 13. The medicament for treating lung cancer asdescribed in claim 9, which is packaged such that about 30 to about 45mg/m² of amrubicin or a pharmaceutically acceptable salt thereof isadministered once a day for 3 days.
 14. The medicament for treating lungcancer as described in claim 9, which is packaged such that about 35 toabout 45 mg/m² of amrubicin or a pharmaceutically acceptable saltthereof is administered once a day for 3 days.
 15. The medicament fortreating lung cancer as described in any of claims 12 to 14, whereinamrubicin or a pharmaceutically acceptable salt thereof is administeredfor continuous 3 days.
 16. The medicament for treating lung cancer asdescribed in claim 1, wherein the other medicament for treating lungcancer is administered in a combined use of about 0.4 times to about 1.0time of the maximum tolerated dose or 0.4 times to about 1.0 time of themaximum administered dose.
 17. The medicament for treating lung canceras described in claim 1, for a patient having a lung cancer, wherein thepatient is one having failed to continue receiving the treatment withthe other medicament for treating lung cancer because of side effects,and wherein the patient is receiving administration of said othermedicament in an amount that will cause reduced side effect.
 18. A useof amrubicin or a pharmaceutically acceptable salt thereof for themanufacture of a medicament for treating lung cancer for a use incombination with another medicament for treating lung cancer.
 19. Amethod for treating a lung cancer which comprises administeringamrubicin or a pharmaceutically acceptable salt thereof and anothermedicament for treating lung cancer.